2.1 The Notch Signaling Pathway
Members of the Notch family encode large transmembrane proteins that play central roles in cell-cell interactions and cell-fate decisions during early development in a number of invertebrate systems. The Notch receptor is part of a highly conserved pathway that enables a variety of cell types to choose between alternative differentiation pathways based on those taken by immediately neighboring cells. This receptor appears to act through a common step that controls the progression of uncommitted cells toward the differentiated state by inhibiting their competence to adopt one of two alternative fates, thereby allowing the cell either to delay differentiation, or in the presence of the appropriate developmental signal, to commit to differentiate along the non-inhibited pathway.
Genetic and molecular studies have led to the identification of a group of genes which define distinct elements of the Notch signaling pathway. While the initial identification of these various elements has came exclusively from Drosophila using genetic tools as the initial guide, subsequent analyses have lead to the identification of homologous proteins in vertebrate species including humans. The molecular relationships between the known Notch pathway elements as well as their subcellular localization are depicted in Artavanis-Tsakonas et al., 1995, Science 268:225-232) and Artvanis-Tsakonas et al., 1999, Science 284:770-776.
2.1.1. Members of the Notch Signaling Pathway
Several members of the Notch signaling pathway have been cloned and sequenced in invertebrate and vertebrate organisms. Non-mammalian Notch genes include those identified in Drosophila (Wharton et al., 1985, Cell 43:567-581); Xenopus (Coffman et al., 1990, Science 249:1438-1441); and zebrafish (Bierkamp et al., 1993, Mech. Dev. 43:87-100). At least four mammalian Notch homologs have been identified (Notch-1, -2, -3, and -4; Weinmaster et al., 1991, Development 113:199-205; Ellisen et al., 1991, Cell 66:523-534; Weinmaster et al., 1992, Development 116:931-941; Franco del Amo et al., 1993, Genomics 15:259-264; Lardelli and Lendahl, 1993, Exp. Cell. Res. 204:364-372; Milner et al., 1994, Blood. 83:2057-62; Lardelli et al., 1994, Mech Dev. 46: 123-136; Uyttendaele et al., 1996, Development 122:2251-9). Other members of the Notch pathway include the ligands Delta and Serrate/Jagged, the cytoplasmic protein Deltex, the transcriptional activator RBP-Jκ, also known as CBF1, downstream targets including but not limited to the Enhancer of Split family of bHLH transcription factors, and, Fringe (Panin et al., 1997, Nature 387:908-912), which acts in the Golgi as a glycosyltransferase enzyme that modifies the epidermal growth factor (EGF) modules of Notch and alters the ability of Notch to bind its ligand Delta. The following non-exhaustive list of articles describes the gene and protein sequences, as well as functional roles, of key members of the Notch signaling pathway:
Invertebrate Ligands: (i) Delta (Kopczynski et al., 1988, Genes Dev. 2:1723-1735; Henrique et al., 1995, Nature 375:787-790; Chitnis et al., 1995, Nature 375:761-766); and (ii) Serrate (Fleming et al., 1990, Genes Dev. 1:2188-2201; Lindsell et al., 1995, Cell 80:909-917; Thomas et al., 1991, Development 111:749-761; Tax et al., 1994, Nature 368:150-154).
Vertebrate Ligands: (i) Serrate (Thomas, 1991, Development 111: 749-761; Lindsell et al., 1995, Cell 80:909-917); and (ii) Delta (Chitnis et al., 1995, Nature 375:761; Henrique et al., 1995, Nature 375:787-790; Bettenhausen et al., 1995, Development 121:2407).
Other Invertebrate Notch Pathway Members: (i) the cytoplasmic protein Deltex (Busseau et al., 1994, Genetics 136:585-596); (ii) the nuclear proteins Mastermind, Hairless, the Enhancer of Split Complex (Smoller et al., 1990, Genes Dev. 4:1688-1700; Bang and Posakony, 1992, Genes Dev. 6:1752-1769; Maier et al., 1992, Mech. Dev. 38:143-156; Delidakis et al., 1991, Genetics 129:803-823; Schrons et al., 1992, Genetics 132:481-503; and Fortini and Artavanis-Tsakonas, 1994, Cell 79:273-282); (iii) Suppressor of Hairless (Furukawa et al., 1991, J. Biol. Chem. 266:23334-23340; Furukawa et al., 1992, Cell 69:1191-1197; and Schweisguth and Posakony, 1992, Cell 69:1199-1212); and (iv) Fringe (Irvine and Wieschaus, 1994, Cell 79:595-606).
Other Vertebrate Notch Pathway Members: (i) RBP-Jκ (Matsunami et al., 1989, Nature 342:934-937; Kawaichi et al., 1992, J. Biol. Chem. 267:4016-4022); (ii) Deltex (Matsunami et al., 1998, Nat. Genet. 19:74-78); (iii) Fringe, including Lunatic, Manic and Radical Fringe (Wu et al., 1996, Science 273:355-358; Moran et al., 1999, Mamm. Genome 10:535-541).
2.1.2. Notch Family Members Encode Surface Receptors that Mediate Inhibitory Signals via the Cytoplasmic Domain
Extensive genetic and molecular studies in Drosophila and C. elegans have shown that the proteins encoded by Notch homologs act as cell surface receptors which can activate inhibitory signal transduction pathways (Greenwald and Rubin, 1992, Cell. 68:271-281; Heitzler and Simpson, 1991, Cell 64:1083-1092; Yochem and Greenwald, 1989, Cell 58:553-63; Fehon et al., 1991, J. Cell Biol. 113:657-669; Rebay et al., 1993, Cell 74:319-329).
Notch signaling is thought to be initiated by interaction with one of the Notch ligands (Delta-1, -2, -3, or Jagged-1 or -2) (Shawber et al., 1996, Developmental Biology 180:370-76; Luo et al., 1997, Molecular and Cellular Biology 17:6057-6067; Henrique et al., 1997, Current Biology 7:661-70; Bettenhausen et al., 1995, Development 121:2407-18; Dunwoodie et al., 1997, Development 124:3065-76). Each of the known ligands is characterized by an extracellular domain containing multiple EGF repeats and a highly conserved DSL domain found in Drosophila, C. elegans, and in vertebrates (Tax et al., 1994, Nature 368:150-154). There is evidence that the ability of particular Notch ligands to induce Notch activation can be modified by the expression of other genes. For example, expression of Fringe prevents activation of Notch by Serrate (Drosophila homolog of Jagged), but enhances Delta activity (Fleming et al., 1997, Development 124:2973-81).
There is considerable evidence that cellular interactions mediated by the extracellular domain modulate signal transduction by the intracellular domain, resulting in regulation of differentiation (Yochem and Greenwald, 1989, Cell 58:553-563; Rebay et al., 1993, Cell 74:319-329). Data indicates that this occurs as a result of binding of the extracellular domain to one of its ligands, followed by a series of proteolytic cleavages which, in turn, leads to release of the intracellular domain of Notch (Struhl and Adachi, 1998, Cell. 93:649-660; Schroeter et al., 1998, Nature 393:382-386). Functional analyses involving the expression of truncated forms of the Notch receptor have indicated that receptor activation depends on the six cdc10/ankyrin repeats in the intracellular domain. Further, Notch activation requires that the cdc10/ankyrin repeats reach the nucleus—possibly after proteolytic cleavage from the remainder of the protein—and participate in transcriptional activation (Struhl and Adachi, 1998, Cell 93:649-660). Deltex and Suppressor of Hairless, whose over-expression results in an apparent activation of the pathway, associate with those repeats. Recent evidence suggests that the proteolytic cleavage step that releases the cdc10/ankyrin repeats for nuclear entry is dependent on Presenilin activity (De Strooper et al., 1999, Nature 398:518-522; Struhl and Greenwald, ibid.:522-525; Ye et al., ibid.:525-529).
The Notch pathway is dependent on protein processing events additional to the step that releases the ankyrin repeats of Notch to the nucleus. The Notch receptor present in the plasma membrane comprises a heterodimer of two Notch proteolytic cleavage products, one comprising an N-terminal fragment including a portion of the extracellular domain, the transmembrane domain and the intracellular domain, and the other including the majority of the extracellular domain (Blaumueller et al., 1997, Cell 0:281-291). The proteolytic cleavage step of Notch to activate the receptor occurs in the Golgi apparatus and is mediated by a furin-like convertase (Logeat et al., 1998, Proc. Natl. Acad. Sci. USA 95:8108-8112). The Notch ligand, Delta, additionally requires cleavage for activation. Delta is thought to be cleaved by ADAM disintegrin metalloprotease Kuzbanian at the cell surface to release a soluble and active form of Delta (Qi et al., 1999, Science 283:91-94).
The intracellular domain of Notch has been shown to act as a constitutively active receptor, because forced expression of this domain prevents myocyte fusion in C2 myoblasts (Kopan et al., 1994, Development 120:2385-2396), blocks muscle conversion of 3T3 cells by MyoD and Myf-5 (Kopan et al., 1994, Development 120:2385-2396), prevents muscle differentiation of DMSO-induced P19 embryonal carcinoma cells, and inhibits neurogenesis while permitting glial differentiation of P19 cells (Nye et al., 1994, Development 120:2421-2430).
The intracellular domain is thought to be transported to the nucleus where it appears to regulate transcription by interacting with a number of molecular targets, including CBF1/RBP-Jκ (Struhl and Adachi, 1998, Cell 93:649-660; Schroeter et al., 1998, Nature 393:382-386 Fortini et al., 1993, Nature 365:555-7). The downstream targets are not completely determined, but RBP-JK is known to activate expression of Hairy Enhancer of Split (HES) which functions as an inhibitor of transcriptional activity (Jarriault et al., 1998, Mol Cell Biol. 18:2230-9). RBP-Jκ, (as stated, also known as CBF1, the homolog of the Drosophila gene Suppressor of Hairless), is a mammalian DNA binding protein involved in the Epstein-Barr virus-induced immortalization of B cells. It has been demonstrated that, at least in cultured cells, Suppressor of Hairless associates with the cdc10/ankyrin repeats in the cytoplasm and translocates into the nucleus upon the interaction of the Notch receptor with its ligand Delta on adjacent cells (Fortini and Artavanis, 1994, Cell 79:273-282). The association of Hairless, a nuclear protein, with Suppressor of Hairless has been documented using the yeast two hybrid system therefore, it is believed that the involvement of Suppressor of Hairless in transcription is modulated by Hairless (Brou et al., 1994, Genes Dev. 8:2491; Knust et al. 1992, Genetics 129:803). It is known that Notch signaling results in the activation of at least certain bHLH genes within the Enhancer of split complex (Delidakis et al., 1991, Genetics 129:803). Mastermind encodes a novel ubiquitous nuclear protein whose relationship to Notch signaling remains unclear but is involved in the Notch pathway as shown by genetic analysis (Smoller et al., 1990, Genes Dev. 4:1688).
There is also evidence that Notch signaling is mediated by an alternative, HES independent pathway, that involves signaling through Deltex and results in repression of E protein activity, e.g. in a B-cell system, it has also been shown that Deltex and not RBP-Jκ, is responsible for inhibiting E47 function (Ordentlich et al., 1998, Mol Cell Biol 18:2230-9). Deltex is a cytoplasmic protein which contains a ring zinc finger and interacts with the ankyrin repeats of Notch (Matsuno et al., 1995, Development 121:2633-2644).
2.1.3. Roles of Notch Family Members
U.S. Pat. No. 5,780,300 describes the roles of Notch proteins in differentiation processes. Briefly, Notch regulates the competence of many different cell types to respond to differentiation/proliferation/apoptosis signals, with the particular cell fates chosen depending upon the developmental history of each cell type and the specific signaling pathways operating within it. In Drosophila and C. elegans, members of the Notchllin-12 family are required at multiple steps during the differentiation of a variety of tissues when specific cell fates are being determined. In C. elegans, the Notch-related genes lin-12 and glp-1 function in a wide variety of cell-cell interactions that result in the inhibition or expression of one or more potential cell fates (Greenwald and Rubin, 1992, Cell 68:271-81; Greenwald et al., 1983, Cell 34:435-444; Austin and Kimble, 1987, Cell 51:589-99; Yochem and Greenwald, 1989, Cell 58:553-563; Wilkinson et al., 1994, Cell 79:1187-1198). One particularly clear example is in the interactions involved in specifying cell fates in the developing vulva, wherein two equivalent multipotent precursors always form one anchor cell (AC) and one ventral uterine precursor (VU) cell (Greenwald and Rubin, 1992, Cell 68:271-81; Greenwald et al., 1983, Cell 34:435-44; Austin and Kimble, 1987, Cell 51:589-99; Yochem and Greenwald, 1989, Cell 58:553-63; Wilkinson et al., 1994, Cell 79:1187-98). If one of the stem cells is eliminated, the remaining cell always becomes an AC; if lin-12 activity is lacking, both become an AC; and if lin-12 activity is elevated, both cells express the VU fate. Further evidence indicates that a relative increase in expression of the ligand for lin-12, lag-2, in the cell committing to AC differentiation induces, via direct cell-cell interaction, an increase in lin-12 activity, which is inhibitory to AC differentiation but permissive for VU differentiation.
In Drosophila, Notch has been shown to be required for appropriate cell-fate decisions in numerous tissues, including the nervous system, eye, mesoderm, ovaries and other areas where multipotent progenitors are making cell-fate decisions (Artavanis-Tsakonas et al., 1999, Science 284:770-776; Go et al., 1998, Development 125:2031-2040; Doherty et al., 1996, Genes Dev. 10:421-434; Artavanis-Tsakonis et al., 1995, Science 268:225-232; Greenwald and Rubin, 1992, Cell 68:271-81; Heitzler and Simpson, 1991, Cell 64:1083-1092; Artavanis-Tsakonas and Simpson, 1991, Trends Genet. 7:403-408; Cagan and Ready, 1989, Genes Dev. 3:1099-1112). In the neurogenic region, for example, the differential expression of Notch appears to mediate a lateral inhibition in which a single cell within a cluster of equivalent cells adopts a neural fate while adjacent cells adopt epidermal fates. Similarly, in embryos with a homozygous null mutation of the Notch gene, all cells in the neurogenic region become neuroblasts and not epidermal precursors.
In Xenopus, the expression of mutant forms of Notch in developing embryos interferes profoundly with normal development (Coffman et al., 1993, Cell 73:659).
Studies of the expression of Notch-1, one of three known vertebrate homologs of Notch, in zebrafish and Xenopus, have shown that the general patterns are similar; with Notch expression associated in general with non-terminally differentiated, proliferative cell populations. Tissues with high expression levels include the developing brain, eye and neural tube (Coffman et al., 1990, Science 249:1438-1441; Bierkamp et al., 1993, Mech. Dev. 43:87-100). While studies in mammals have shown the expression of the corresponding Notch homologs to begin later in development, the proteins are expressed in dynamic patterns in tissues undergoing cell fate determination or rapid proliferation (Weinmaster et al., 1991, Development 113:199-205; Reaume et al., 1992, Dev. Biol. 154:377-387; Stifani et al., 1992, Nature Genet. 2:119-127; Weinmaster et al., 1992, Development 116:931-941; Kopan et al., 1993, J. Cell Biol. 121:631-641; Lardelli et al., 1993, Exp. Cell Res. 204:364-372; Lardelli et al., 1994, Mech. Dev. 46:123-136; Henrique et al., 1995, Nature 375:787-790; Horvitz et al., 1991, Nature 351:535-541; Franco del Amo et al., 1992, Development 115:737-744). Among the tissues in which mammalian Notch homologs are first expressed are the pre-somitic mesoderm and the developing neuroepithelium of the embryo. In the pre-somitic mesoderm, expression of Notch-1 is seen in all of the migrated mesoderm, and a particularly dense band is seen at the anterior edge of pre-somitic mesoderm. This expression has been shown to decrease once the somites have formed, indicating a role for Notch in the differentiation of somatic precursor cells (Reaume et al., 1992, Dev. Biol. 154:377-387; Horvitz et al., 1991, Nature 351:535-541). Similar expression patterns are seen for mouse Delta (Simske et al., 1995, Nature 375: 142-145).
Within the developing mammalian nervous system, expression patterns of Notch homolog have been shown to be prominent in particular regions of the ventricular zone of the spinal cord, as well as in components of the peripheral nervous system, in an overlapping but non-identical pattern. Notch expression in the nervous system appears to be limited to regions of cellular proliferation, and is absent from nearby populations of recently differentiated cells (Weinmster et al., 1991, Development 113:199-205; Reaume et al., 1992, Dev. Biol. 154:377-387; Weinmaster et al., 1992, Development 116:931-941; Kopan et al., 1993, J. Cell Biol. 121:631-641; Lardelli et al., 1993, Exp. Cell Res. 204:364-372; Lardelli et al., 1994, Mech. Dev. 46:123-136; Henrique et al., 1995, Nature 375:787-790; Horvitz et al., 1991, Nature 351:535-541). A rat Notch ligand is also expressed within the developing spinal cord, in distinct bands of the ventricular zone that overlap with the expression domains of the Notch genes. The spatio-temporal expression pattern of this ligand correlates well with the patterns of cells committing to spinal cord neuronal fates, which demonstrates the usefulness of Notch as a marker of populations of cells for neuronal fates (Henrique et al., 1995, Nature 375:787-790). This has also been suggested for vertebrate Delta homologs, whose expression domains also overlap with those of Notch-1 (Larsson et al., 1994, Genomics 24:253-258; Fortini et al., 1993, Nature 365:555-557; Simske et al., 1995, Nature 375: 142-145). In the cases of the Xenopus and chicken homologs, Delta is actually expressed only in scattered cells within the Notch-1 expression domain, as would be expected from the lateral specification model, and these patterns “foreshadow” future patterns of neuronal differentiation (Larsson et al., 1994, Genomics 24:253-258; Fortini et al., 1993, Nature 365:555-557).
Other vertebrate studies of particular interest have focused on the expression of Notch homologs in developing sensory structures, including the retina, hair follicles and tooth buds. In the case of the Xenopus retina, Notch-1 is expressed in the undifferentiated cells of the central marginal zone and central retina (Coffman et al., 1990, Science 249:1439-1441; Mango et al., 1991, Nature 352:811-815). Studies in the rat have also demonstrated an association of Notch-1 with differentiating cells in the developing retina and have been interpreted to suggest that Notch-1 plays a role in successive cell fate choices in this tissue (Lyman et al., 1993, Proc. Natl. Acad. Sci. USA 90:10395-10399).
A detailed analysis of mouse Notch-1 expression in the regenerating matrix cells of hair follicles was undertaken to examine the potential participation of Notch proteins in epithelial/mesenchymal inductive interactions (Franco del Amo et al., 1992, Development 115:737-744). Such a role had originally been suggested for Notch-1 based on its expression in rat whiskers and tooth buds (Weinmaster et al., 1991, Development 113:199-205). Notch-1 expression was instead found to be limited to subsets of non-mitotic, differentiating cells that are not subject to epithelial/mesenchymal interactions, a finding that is consistent with Notch expression elsewhere.
The human homolog of Notch-1 (TAN-1) was initially cloned from a T-cell leukemia with a translocation involving this gene and subsequently found in a variety of adult tissues, but in greatest amounts in thymus and lymph node (Ellisen et al., 1991, Cell 66:649-661; Zhong et al., 1997, Development 124:1887-1897; Vargesson et al., 1998, Mech Dev. 77:197-9; Lewis et al., 1998, Mech Dev. 78:159-163; Lindsell et al., 1996, Mol. Cell. Neurosci. 8:14-27; Hasserjian et al., 1996, Blood. 88:970-976). A homolog of Notch/TAN-1 is expressed in human CD34+ hematopoietic precursors (Milner et al., 1994, Blood 83:2057-2062) as well as CD34− bone marrow cells (Milner et al., 1994, Blood 83:2057-2062; Varnum-Finney et al., 1998, Blood 91:4084-4091). Subsequent studies demonstrated widespread expression of Notch-1 and Notch-2 protein during hematopoietic development, as well as the Notch ligand, Jagged-1, in hematopoietic stroma (Varnum-Finney et al., 1998, Blood 91:4084-4091; Li et al., 1998, Immunity 8:43-55). The preferential expression of vertebrate Notch homologs in tissues undergoing cellular proliferation and differentiation suggests that these molecules are involved in mediating cell-fate decisions in vertebrates as they do in invertebrates. This persistence in tissues that are mitotically active also suggests that Notch may be involved in regulating cell proliferation. Consistent with this notion is the oncogenic phenotype associated with deregulated expression of the cytoplasmic domain of Notch-1 and, in mice, of the Notch-related int-3 locus which is a common integration site for mouse mammary tumor viruses in virus-induced tumors (Jhappan et al., 1992, Genes Dev. 6:345-355; Robbins et al., 1992, J. Virol. 66:2594-2599).
Additional studies of human Notch-1 and Notch-2 expression have been performed on adult tissue sections including both normal and neoplastic cervical and colon tissue. Notch-1 and Notch-2 appear to be expressed in overlapping patterns in differentiating populations of cells within squamous epithelia of normal tissues that have been examined and are clearly not expressed in normal columnar epithelia, except in some of the precursor cells. Both proteins are expressed in neoplasias, in cases ranging from relatively benign squamous metaplasias to cancerous invasive adenocarcinomas in which columnar epithelia are replaced by these tumors (Gray et al., 1999, Am. J. Pathol. 154:785-794; Zagouras et al., 1995, Proc. Natl. Acad. Sci. USA 92:6414-6418).
2.1.4. Notch Functions in Hematopoiesis
Evidence of Notch-1 mRNA expression in human CD34+ precursors has led to speculation for a role for Notch signaling in hematopoiesis (Milner et al., 1994, Blood 3:2057-62). This is further supported by the demonstration that Notch-1 and -2 proteins are present in hematopoietic precursors and, in higher amounts, in T cells, B cells, and monocytes, and by the demonstration of Jagged-1 protein in hematopoietic stroma (Ohishi et al., 2000, Blood 95:2847-2854; Varnum-Finney et al., 1998, Blood 91:4084-91; Li et al., 1998, Immunity 8:43-55).
The clearest evidence for a physiologic role of Notch signaling has come from studies of T cell development which showed that activated Notch-1 inhibited B cell maturation but permitted T cell maturation (Pui et al., 1999, Immunity 11:299-308). In contrast, inactivation of Notch-1 or inhibition of Notch-mediated signaling by knocking out HES-1 inhibited T cell development but permitted B cell maturation (Radtke et al., 1999, Immunity 10: 47-58; Tomita et al., 1999, Genes Dev. 13:1203-10). These opposing effects of Notch-1 on B and T cell development raise the possibility that Notch-1 regulates fate decisions by a common lymphoid progenitor cell.
Other studies in transgenic mice have shown that activated Notch-1 affects the proportion of cells assuming a CD4 vs. CD8 phenotype as well as an αβ vs. γΔ cell-fate (Robey et al., 1996, Cell 87:483-92; Washburn et al., 1997, Cell 88:833-43). Although this may reflect an effect on fate decisions by a common precursor, more recent studies have suggested that these effects may result from an anti-apoptotic effect of Notch-1 that enables the survival of differentiating T cells that would otherwise die (Deftos et al., 1998, Immunity 9:777-86; Jehn et al., 1999, J. Immunol. 162:635-8).
Evidence supporting a critical role for Notch signaling in myelopoiesis is less clear. In vivo studies involving overexpression or inactivation of Notch-1 have not identified significant effects of Notch-1 signaling on the development of mature myeloid elements, despite profound effects on T and B cell development (Pui et al., 1999, Immunity 11:299-308; Radtke et al., 1999, Immunity 10:547-58). However, in vitro studies have demonstrated effects of constitutively active Notch-1 forms on myelopoiesis. Constitutive overexpression of an activated form of Notch-1 inhibited G-CSF-induced granulocytic differentiation of murine 32D cells (Milner et al., 1996, Proc Natl Acad Sci U.S.A. 93:13014-9). More recent studies suggest that overexpression of the constitutively active intracellular domain of Notch-1 inhibits the differentiation of isolated murine hematopoietic precursors and enhances the generation of early precursor cells, including in vivo repopulating cells (Milner et al., 1996, Proc. Natl. Acad. Sci. U.S.A. 93:13014-13019; Bigas et al., 1998, J. Mol. Cell. Biol. 18:2324-2333). Thus, the lack of identifiable effects of Notch-1 on the in vivo generation of mature myeloid elements may result from compensatory effects due to other factors such as cytokines which may mask the effects of Notch activation in less mature precursors.
Studies have also shown that the differentiation of isolated hematopoietic precursor cells can be inhibited by ligand-induced Notch signaling. Coculture of murine marrow precursor cells (lin-Sca-1+ c-kit+) with 3T3 cells expressing human Jagged-1 led to a 2 to 3 fold increase in the formation of primitive precursor cell populations (Varnum-Finney et al., 1998, Blood 91:4084-4991; Jones et al., 1998, Blood 92:1505-11). Incubation of sorted precursors with beads coated with the purified extracellular domain of human Jagged-1 also led to enhanced generation of precursor cells (Varnum-Finney et al., 1998, Blood 91:4084-91).
In a study of human CD34+ cells, expression of the intracellular domain of Notch-1 or exposure to cells that overexpressed Jagged-2 also led to enhanced generation of precursor cells and prolonged maintenance of CD34 expression (Carlesso et al., 1999, Blood 93:838-48). In another study, the effects of Jagged-1-expressing cells on CD34+ cells were influenced by the cytokines present in the cultures; in the absence of added growth factors, the interaction with cell-bound Jagged-1 led to maintenance of CD34+ cells in a non-proliferating, undifferentiated state, whereas the addition of c-kit ligand led to a 2-fold increase in erythroid colony-forming cells (Walker et al., 1999, Stem Cells 17:162-71).
Studies of more mature myeloid elements have also indicated a potential role for Notch signaling in regulating their cell-fate decisions. In those studies, immobilized, truncated Delta-1 inhibited the differentiation of CD14+monocytes into macrophages and induced apoptosis in the presence of specific cytokines (Ohishi et al., 2000, Blood 95:2847-2854). Further, ligand-induced Notch signaling is permissive for differentiation of monocytes into dendritic cells in the context of appropriate cytokine stimulation. Thus, as observed in other developing systems, Notch signaling appears to inhibit differentiation along a particular pathway, allowing cells to remain undifferentiated or to differentiate along the uninhibited, default pathway.
Notch signaling has been shown to play a central role in cell fate decisions in numerous developmental systems. The evolutionarily conserved Notch transmembrane receptors are known to play roles in differentiation, proliferation, and apoptotic events. In general, Notch signaling inhibits differentiation along a particular pathway, allowing the cell to remain undifferentiated or differentiate along an alternate pathway in response to specific environmental cues. Notch signaling is induced following receptor ligand interaction, causing proteolytic cleavage and release of an active intracellular domain which is transported to the nucleus and interacts with a number of downstream targets, including the transcriptional regulator, RBP-Jκ. At present, four paralogs of the Notch gene have been identified in vertebrates (Notch-1-4). The ligands for Notch are also transmembrane proteins and include Jagged-1 and -2, and Delta-1, -2, and -3. Evidence of expression of Notch-1 mRNA in human CD34+ precursors has led to speculation for a role for Notch signaling in hematopoiesis. Further studies have demonstrated Notch-1 and -2 protein in hematopoietic precursors and, in higher amounts, in T cells, B cells, and monocytes, as well as showing Jagged-1 to be expressed in hematopoietic stroma. The clearest evidence for a physiologic role of Notch signaling has come from studies of T cell development where Notch-1 mediated signaling is required for T cell development and affects CD4/CD8 and αβ/γΔ cell fate decisions, and constitutively active forms of Notch-1 induce T cell lymphomas. In addition, overexpression of a constitutively active Notch-1 form inhibits B cell maturation, suggesting that Notch-1 may regulate fate decisions by a common lymphoid progenitor cell. Evidence supporting a critical role for Notch signaling in myelopoiesis is less clear. Constitutive overexpression of an activated Notch-1 form inhibits G-CSF-induced granulocytic differentiation of 32D cells, and the differentiation of isolated hematopoietic precursors. The differentiation of precursor cells is also inhibited by ligand-induced Notch signaling. Coculture of murine marrow precursor cells (sca-1+ lin− c-kit+) with a 3T3 cell layer that expresses human Jagged-1 or incubating sorted precursors with beads coated with the purified extracellular domain of human Jagged-1 leads to a 2-3 fold increase in the formation of primitive precursor cell populations. Immobilized, truncated forms of the Notch ligand, Delta-1, were found to inhibit the differentiation of isolated precursors, allowing a substantial increase in the number of sca-1+lin− cells.
2.2 Cellular Differentiation During Development
The developmental processes that govern the ontogeny of multicellular organisms, including humans, depend on the interplay between signaling pathways, which gradually narrow the developmental potential of cells from the original totipotent stem cell to the terminally differentiated mature cell, which performs a specialized function, such as a heart cell or a nerve cell.
The fertilized egg is the cell from which all other cell lineages derive, i.e., the ultimate stem cell. As development proceeds, early embryonic cells respond to growth and differentiation signals which gradually narrow the cells' developmental potential, until the cells reach developmental maturity, i.e., are terminally differentiated. These terminally differentiated cells have specialized functions and characteristics, and represent the last step in a multi-step process of precursor cell differentiation into a particular cell.
The transition from one step to the next in cell differentiation is governed by specific biochemical mechanisms which gradually control the progression until maturity is reached. It is clear that the differentiation of tissues and cells is a gradual process which follows specific steps until a terminally differentiated state is reached.
Gastrulation, the morphogenic movement of the early embryonic cell mass, results in the formation of three distinct germ cell layers, the ectoderm, the mesoderm, and the endoderm. As cells in each germ cell layer respond to various developmental signals, specific organs are generated which are composed of specific differentiated cells. For example, the epidermis and the nervous system develop from ectoderm-derived cells, the respiratory system and the digestive tract are developed from endoderm-derived cells, and mesoderm-derived cells develop into the connective tissues, the hematopoietic system, the urogenital system, muscle, and parts of most internal organs.
The neural crest derives from the ectoderm and is the cell mass from which an extraordinary large and complex number of differentiated cell types are produced, including the peripheral nervous system, pigment cells, adrenal medulla and certain areas of the head cartilage.
The pluripotentiality of neural crest cells is well established (LeDouarin et al., 1975, Proc. Natl. Acad. Sci. USA 72:728-732). A single neural crest cell can differentiate into several different cell types.
The epidermis consists of several cellular layers which define a differentiation lineage starting from the undifferentiated, mitotically active basal cells to the terminally differentiated non-dividing keratinocytes.
The endoderm is the source of the tissues that line two tubes within the adult body. The digestive tube extends throughout the length of the body. The digestive tube gives rise not only to the digestive tract but also to, for example, the liver, the gallbladder and the pancreas. The second tube, the respiratory tube, forms the lungs and part of the pharynx. The pharynx gives rise to the tonsils, thyroid, thymus, and parathyroid glands.
The genesis of the mesoderm which has also been referred to as the mesengenic process gives rise to a very large number of internal tissues which cover all the organs between the ectodermal wall and the digestive and respiratory tubes.
Embryonic development produces the fully formed organism. The morphologic processes that define the cellular boundaries of each organ include not only proliferation and differentiation, but also apoptosis (programmed cell death). For example, in the nervous system, approximately 50% of neurons undergo programmed cell death during embryogenesis.
In the juvenile or adult individual, the maintenance of tissues, whether during normal life or in response to injury and disease, depends on the replenishing of the organs from precursor cells that are capable of responding to specific developmental signals.
The best known example of adult cell renewal via the differentiation of immature cells is the hematopoietic system. Here, developmentally immature precursors (hematopoietic stem and progenitor cells) respond to molecular signals to gradually form the varied blood and lymphoid cell types.
During hematopoietic development, the progeny of pluripotent stem cells progressively lose their proliferative potential and capacity for self-renewal, and display greater commitment to a given differentiation pathway. The factors that regulate this commitment to the various hematopoietic lineages are not understood, but are thought to include stochastic processes and interactions with soluble and cell-bound cytokines (Fairbairn et al., 1993, Cell 4:823-32; Ogawa, 1993, Blood 81:2844-53; Metcalf, 1989, Nature 339:27-30; Metcalf, 1993, Blood. 82:3515-23; Goldsmith et al., 1998, Proc. Natl. Acad. Sci. USA. 95:7006-11; Socolovsky et al., 1997, J. Biol. Chem. 272:14009-12).
While the hematopoietic system is the best understood self renewing adult cellular system it is believed that most, perhaps all, adult organs harbor precursor cells that under the right circumstances, can be triggered to replenish the adult tissue. For example, the pluripotentiality of neural crest cells has been described above. The adult gut contains immature precursors which replenish the differentiated tissue. Liver has the capacity to regenerate because it contains hepatic immature precursors; skin renews itself, etc. Through the mesengenic process, most mesodermal derivatives are continuously replenished by the differentiation of precursors. Such repair recapitulates the embryonic lineages and entails differentiation paths which involve pluripotent progenitor cells.
Mesenchymal progenitor cells are pluripotent cells that respond to specific signals and adopt specific lineages. For example, in response to bone morphogenic factors, mesenchymal progenitor cells adopt a bone forming lineage. For example, in response to injury, mesodermal progenitor cells can migrate to the appropriate site, multiply and react to local differentiation factors, consequently adopting a distinct differentiation path.
It has been suggested that the reason that only a limited tissue repair is observed in adults is because there are too few progenitor cells which can adopt specific differentiation lineages. It is clear that if these cells can be expanded by immortalizing them in culture, then tissue repair could be facilitated by transplantation of the cultured cells. However, diploid cells generally have a limited proliferative capacity in vitro. Following initial culturing, the cells undergo a series of rapid cycling, which slows down until the population undergoes a growth arrest, which is a result of a block at the G1/S or G2/M phases of mitosis (Derventz et al., 1996, Anticancer Res. 16:2901-2910). For example, after a limited number of divisions, human fibroblasts enter a nonreplicative state as a result of cellular senescence. When certain viral oncogenes are expressed in the fibroblasts, the replicative life span is extended, but the cells still enter a nonreplicative state termed a “crisis” state (Wei and Sedivy, 1999, Exp Cell Res 253:519-522). The number of cell cycles a cell undergoes before reaching the growth arrest phase depends on the cell type; for human cells, the number is generally between 30 and 60 (Derventz et al., 1996, Anticancer Res. 16:2901-2910 and reference cited therein). Therefore, the process of immortalizing pluripotent or multipotent cells, such as stem or progenitor cells of a desired type, ex vivo would give rise to more rapid proliferation of the desired cell type and allow for more rapid treatment injuries or traumas. Additionally, the ability would give rise to the potential for treating many human diseases and could circumvent tissue rejection without the need for immunosuppressive agents.